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• W1977917516 abstract "Point and deletion mutants of moesin were examined for F-actin binding by blot overlay and co-sedimentation, and for intra- and intermolecular interactions with N- and C-terminal domains with yeast two-hybrid and in vitro binding assays. Wild-type moesin molecules interact poorly with F-actin and each other, and bind neither C- nor N-terminal fragments. Interaction with F-actin is strongly enhanced by replacement of Thr558 with aspartate (T558D), by deletion of 11 N-terminal residues (DelN11), by deletion of the entire N-terminal membrane-binding domain of both wild type and T558D mutant molecules, and by exposure to phosphatidylinositol 4,5-diphosphate. Activation of F-actin binding is accompanied by changes in inter- and intramolecular domain interactions. The T558D mutation renders moesin capable of binding wild type but not mutated (T558D) C-terminal or wild type N-terminal fragments. The interaction between the latter two is prevented. DelN11 truncation enables binding of wild type N and C domain fragments. These changes suggest that the T558D mutation, mimicking phosphorylation of Thr558, promotes F-actin binding by disruption of interdomain interactions between N and C domains and exposure of the high affinity F-actin binding site in the C-terminal domain. Oscillation between activated and resting state could thus provide the structural basis for transient interactions between moesin and the actin cytoskeleton in protruding and retracting microextensions. Point and deletion mutants of moesin were examined for F-actin binding by blot overlay and co-sedimentation, and for intra- and intermolecular interactions with N- and C-terminal domains with yeast two-hybrid and in vitro binding assays. Wild-type moesin molecules interact poorly with F-actin and each other, and bind neither C- nor N-terminal fragments. Interaction with F-actin is strongly enhanced by replacement of Thr558 with aspartate (T558D), by deletion of 11 N-terminal residues (DelN11), by deletion of the entire N-terminal membrane-binding domain of both wild type and T558D mutant molecules, and by exposure to phosphatidylinositol 4,5-diphosphate. Activation of F-actin binding is accompanied by changes in inter- and intramolecular domain interactions. The T558D mutation renders moesin capable of binding wild type but not mutated (T558D) C-terminal or wild type N-terminal fragments. The interaction between the latter two is prevented. DelN11 truncation enables binding of wild type N and C domain fragments. These changes suggest that the T558D mutation, mimicking phosphorylation of Thr558, promotes F-actin binding by disruption of interdomain interactions between N and C domains and exposure of the high affinity F-actin binding site in the C-terminal domain. Oscillation between activated and resting state could thus provide the structural basis for transient interactions between moesin and the actin cytoskeleton in protruding and retracting microextensions. Moesin is one of several closely related and widely expressed proteins (1Lankes W.T. Furthmayr H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8279-8301Crossref Scopus (210) Google Scholar, 2Funayama N. Nagafuchi A. Sato N. Tsukita S. Tsukita S. J. Cell Biol. 1991; 115: 1039-1048Crossref PubMed Scopus (137) Google Scholar, 3Gould K.L. Bretscher A. Esch F.S. Hunter T. EMBO J. 1989; 8: 4133-4142Crossref PubMed Scopus (207) Google Scholar). They include ezrin and radixin and share ∼75% overall sequence identity and regions, denoted A–H, of even higher structural conservation (4Lankes W. Schwartz-Albiez R. Furthmayr H. Biochim. Biophys. Acta. 1993; 1216: 479-482Crossref PubMed Scopus (29) Google Scholar). A common secondary structure is predicted to consist of a 320-residue globular N-terminal domain, a central ∼200-residue predominantly α-helical region, and a ∼50-residue highly charged C-terminal domain. Based on a large body of evidence, these proteins bind to components of the cell membrane with the N-terminal domain (5Amieva M.R. Litman P. Huang L. Ichimaru E. Furthmayr H. J. Cell Sci. 1998; 112: 111-125Google Scholar, 6Algrain M. Turunen O. Vaheri A. Louvard D. Arpin M. J. Cell Biol. 1993; 120: 129-139Crossref PubMed Scopus (372) Google Scholar, 7Amieva M.R. Wilgenbus K.K. Furthmayr H. Exp. Cell Res. 1994; 210: 140-144Crossref PubMed Scopus (61) Google Scholar, 8Amieva M.R. Furthmayr H. Exp. Cell Res. 1995; 219: 180-196Crossref PubMed Scopus (132) Google Scholar, 9Hanzel D. Reggio H. Bretscher A. Forte J.G. Mangeat P. EMBO J. 1991; 10: 2363-2373Crossref PubMed Scopus (151) Google Scholar, 10Berryman M. Franck Z. Bretscher A. J. Cell Sci. 1993; 105: 1025-1043Crossref PubMed Google Scholar, 11Sato N. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Sci. 1992; 103: 131-143PubMed Google Scholar). Candidate membrane-binding targets include CD44; intercellular adhesion molecules 1, 2, and 3 (12Yonemura S. Hirao M. Doi Y. Takahashi N. Kondo T. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 885-895Crossref PubMed Scopus (503) Google Scholar, 13Serrador J.M. Alonso-Lebrero J.L. del Pozo M.A. Furthmayr H. Schwartz-Albiez R. Calvo J. Lozano F. Sanchez-Madrid F. J. Cell Biol. 1997; 138: 1409-1423Crossref PubMed Scopus (200) Google Scholar, 14Serrador J.M. Nieto M. Alonso-Lebrero J.L. del Pozo M.A. Furthmayr H. Schwartz-Albiez R. Gonzalez-Amaro R. Sanchez-Mateos P. Sanchez-Madrid F. Blood. 1998; 91: 4632-4644Crossref PubMed Google Scholar); phosphatidylinositol 4,5-disphosphate (15Niggli V. Andreoli C. Roy C. Mangeat P. FEBS Lett. 1995; 376: 172-176Crossref PubMed Scopus (162) Google Scholar); cyclic AMP-dependent protein kinase (16Dransfield D.T. Bradford A.J. Smith J. Martin M. Roy C. Mangeat P.H. Goldenring J.R. EMBO J. 1997; 16: 35-43Crossref PubMed Scopus (263) Google Scholar); and intermediary adapters of the NHERF family of proteins (17Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (512) Google Scholar, 18Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (400) Google Scholar). The C-terminal domain contains a major high affinity actin binding site, presumed to be located in region H, a sequence that is identical in ezrin and radixin (19Turunen O. Wahlstrom T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (343) Google Scholar, 20Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar, 21Yao X. Cheng L. Forte J.G. J. Biol. Chem. 1996; 271: 7224-7229Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 22Edwards K.A. Demsky M. Montague R.A. Weymouth N. Kiehart D.P. Dev. Biol. 1997; 191: 103-117Crossref PubMed Scopus (222) Google Scholar, 23Huang L. Ichimaru E. Pestonjamasp K. Cui X. Nakamura H. Lo G.Y.H. Lin F.I.K. Luna E.J. Furthmayr H. Biochem. Biophys. Res. Commun. 1998; 248: 548-553Crossref PubMed Scopus (37) Google Scholar). This sequence differs in the tumor suppressor merlin, and while F-actin binds to the moesin sequence, it does not bind to merlin under the same conditions (23Huang L. Ichimaru E. Pestonjamasp K. Cui X. Nakamura H. Lo G.Y.H. Lin F.I.K. Luna E.J. Furthmayr H. Biochem. Biophys. Res. Commun. 1998; 248: 548-553Crossref PubMed Scopus (37) Google Scholar). Moesin and related proteins are required for the formation of membrane microextensions (5Amieva M.R. Litman P. Huang L. Ichimaru E. Furthmayr H. J. Cell Sci. 1998; 112: 111-125Google Scholar, 7Amieva M.R. Wilgenbus K.K. Furthmayr H. Exp. Cell Res. 1994; 210: 140-144Crossref PubMed Scopus (61) Google Scholar, 8Amieva M.R. Furthmayr H. Exp. Cell Res. 1995; 219: 180-196Crossref PubMed Scopus (132) Google Scholar, 24Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (308) Google Scholar) and for the GTPase-mediated formation of stress fibers, focal adhesion complexes, and microextensions (25Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3786) Google Scholar, 26Ridley A.J. Paterson H.F. Johnston C.J. Diekman D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3040) Google Scholar, 27Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (265) Google Scholar). Interactions of proteins in the cell cortex underlie a variety of cellular responses, such as changes in cell shape, adhesion, movement, and signal transduction. The function of moesin in these dynamic processes at the membrane/cytoskeletal interface very likely depends on regulation of its binding activities (28Nakamura F. Amieva M.R. Furthmayr H. J. Biol. Chem. 1995; 270: 31377-31385Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 29Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (721) Google Scholar). Several proposals have been made, but it has not been firmly established as yet how the linkage function of moesin operates and how it is regulated in cells. In vitro experiments have demonstrated homotypic and heterotypic associations between ezrin and moesin and small amounts of dimeric molecules have been isolated from cell lysates by immunoprecipitation (30Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (372) Google Scholar, 31Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar). This, combined with the fact that isolated N and C domains of these proteins interact, has led to the suggestion that oligomers are functionally important and that they are formed by disruption of an intramolecular interaction between N-terminal and C-terminal domains (30Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (372) Google Scholar, 32Martin M. Andreoli C. Sahuquet A. Montcourrier P. Algrain M. Mangeat P. J. Cell Biol. 1995; 128: 1081-1093Crossref PubMed Scopus (120) Google Scholar, 33Magendantz H. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Consistent with this idea is that the binding sites mediating this head-to-tail association are masked in the full-length protein, when tested for interaction of full-length molecules with either C or N domains (30Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (372) Google Scholar). A second proposal has been that phosphorylation activates binding functions of moesin. In human platelets, for instance, thrombin activation leads to a rapid, but transient, increase in the phosphorylation of a single threonine, Thr558, in the C-terminal domain and near the presumptive location of the F-actin binding site (20Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar, 28Nakamura F. Amieva M.R. Furthmayr H. J. Biol. Chem. 1995; 270: 31377-31385Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). This residue is conserved in moesin, ezrin, and radixin, and it is modified also in RAW macrophages (34Nakamura F. Amieva M.R. Hirota C. Mizuno Y. Furthmayr H. Biochem. Biophys. Res. Commun. 1996; 226: 650-656Crossref PubMed Scopus (35) Google Scholar), Swiss3T3 cells (29Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (721) Google Scholar), 1F. Nakamura and H. Furthmayr, unpublished observations.1F. Nakamura and H. Furthmayr, unpublished observations. NIH3T3 cells, 2L. Huang and H. Furthmayr, unpublished observations.2L. Huang and H. Furthmayr, unpublished observations. and RB2H3 mast cells. 3E. Ichimaru and H. Furthmayr, unpublished observations.3E. Ichimaru and H. Furthmayr, unpublished observations. Phosphorylation at this site is regulated by the activity of Rho, and Rho-associated kinase phosphorylates two residues in the C-terminal region, one of which is Thr558 (29Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (721) Google Scholar). Thr558 can also be phosphorylated by θ-phosphokinase C (35Pietromonaco S.F. Simons P.C. Altman A. Elias L. J. Biol. Chem. 1998; 273: 7594-7603Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Phosphorylation of this conserved amino acid by Rho-associated kinase prevents interaction of the phosphorylated C domain with the N-terminal domain (29Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (721) Google Scholar) but does not appear to influence binding of the isolated C domain to F-actin. Other than Thr558, the role of phosphorylation of specific tyrosine, serine, or other threonine residues in response to stimulation by growth factors, histamine, or lysophosphatidic acid has not been established (36Urushidani T. Hanzel D.K. Forte J.G. Am. J. Physiol. 1989; 256: G1070-G1080Crossref PubMed Google Scholar, 37Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 38Krieg J. Hunter T. J. Biol. Chem. 1992; 267: 19258-19265Abstract Full Text PDF PubMed Google Scholar, 39Fazioli F. Wong W.T. Ullrich S.J. Sakaguchi K. Appella E. Di Fiore P.P. Oncogene. 1993; 8: 1335-1345PubMed Google Scholar, 40Shaw R.J. Henry M. Solomon F. Jack T. Mol. Biol. Cell. 1998; 9: 403-419Crossref PubMed Scopus (159) Google Scholar). Phosphorylation of Thr558 of moesin is of significance in human platelets, because it is modulated by physiological activation, it can be manipulated by protein kinase and phosphatase inhibitors, and it is accompanied by changes in moesin distribution and platelet morphology (28Nakamura F. Amieva M.R. Furthmayr H. J. Biol. Chem. 1995; 270: 31377-31385Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). We believe that phosphorylation of Thr558can be mimicked by replacement of threonine with aspartate, and the present study was undertaken to test the hypothesis that this modification activates the F-actin binding function of moesin. We show that Thr558 substitution greatly enhances F-actin binding activity of moesin by comparing the actin filament binding properties of wild type moesin and a series of mutants by blot overlay and co-sedimentation assays and by examining in detail interdomain and intermolecular interactions by in vitro assays and with the yeast two-hybrid system in vivo. Furthermore, we present several lines of strong evidence that this activation step is linked to the disruption of interactions between the N- and C-terminal domains. This in turn changes the overall conformation of the protein and exposes the high affinity binding site in the C-terminal domain. Mutations of moesin Thr558within the sequence KYKT558L were introduced into moesin cDNA by PCR 4The abbreviations used are: PCR, polymerase chain reaction; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-diphosphate; kb, kilobase pair(s)using oligonucleotides as primers that specify both the desired mutation and carry a convenient restriction site to facilitate subcloning and isolation of mutant clones. The following oligonucleotides were synthesized: A, GEX5′(+) (5′-CCAAAATCGGATCTGGTTCCG-3′); B, MSN T558D Bgl2(−) (5′-CCGGATCTGGCGCAGATCTTTGTATTTGTCTCGGCC-3′); C, MSN T558D Bgl2(+) (5′-GGCCGAGACAAATACAAAGATCTGCGCCAGATCCGG-3′); D, GEX3′Aat2(−) (5′-CTCTCAAGGATCTTACCGCTG-3′); E, MSN T558AHindII(−) (5′-CCGGATCTGGCGCAAAGCTTTGTATTTGTCTCGGC-3′); F, MSN T558AHindII(+) (5′-GCCGAGACAAATACAAAGCTTTGCGCCAGATCCGG-3′); G, MSN3′SalI(−) (5′-AATTTTAGGTCAGTCGACATCCCTGGAG-3′); H, MSN DK5 PvuI(−) (5′-GCGCAGGGTCTTGTATCGATCGCGGCCCAGTCGCATGT-3′); I, MSN DK5PvuI(+) (5′-CGAGACAAATACAAGACGATCGCCAGATCCGGCAG-3′). To substitute Thr558 with Asp (D), two PCR reactions were performed using pGhuMo (pGEX-KG-human moesin, or GST-MSN (20Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar)) plasmid DNA as template with oligonucleotides A and B as primers in reaction 1, and C and D in reaction 2. The product of reaction 1 was digested with BglII yielding a 1.1-kb fragment, and the product of reaction 2 gave a 0.5-kb fragment by digestion withBglII and AatII. The two fragments were then subcloned by three-way ligation into pGhuMo that had been predigested with BglII and AatII to delete the C-terminal portion of moesin. This resulted in pKG-MSNT558D (GST-MSNT558D). To substitute Thr558 with Ala, two PCRs were performed. In reaction 1, pGhuMo served as template, and oligonucleotides A and E served as primers. In reaction 2, UIII (Ref. 3Gould K.L. Bretscher A. Esch F.S. Hunter T. EMBO J. 1989; 8: 4133-4142Crossref PubMed Scopus (207) Google Scholar; human moesin in pBSSK+) served as template, and F and G served as primers. Reaction 1 yielded a 1.1-kb BglII–HindIII fragment, and reaction 2 yielded a 0.5-kbHindIII–SalI fragment. These two fragments were subcloned by three-way ligation intoBglII/SalI-predigested pGhuMo to delete the C-terminal portion of moesin and to generate pKG-MSNT558A (GST-MSNT558A). To delete KYKT558L, two PCRs were performed with oligonucleotides A and H as primers in reaction 1 and I and D in reaction 2 using pGhuMo as template. The first reaction yielded a 1.1-kb BglII–PvuI fragment, and the second yielded a 0.5-kb PvuI–AatII fragment. The two fragments were subcloned by three-way ligation intoBglII/AatII-predigested pGhuMo to result in pKG-MSNΔK5 (GST-MSNΔK5). pKG-MSNT558A was digested with HindIII to delete the C-terminal 19 codons and recircularized to generate pKG-MSNΔC19 (GST-MSNΔC19). pKG-MSNT558D was digested with BglII to delete encoding residues 198–558 and recircularized to generate pKG-MSNΔ(L198-T558) (GST-MSNΔm). pKG-MSNc (GST-moesin C-terminal region residues 404–577) was constructed by inserting the EcoRI–XhoI fragment from MSNc/pCR3 (see below) into pGEX-KG. pKG-MSNn (GST-moesin N-terminal region residues 1–310) was generated by subcloning the EcoRI–SalI fragment from pAS1-MSNn (see below) into pGEX-KG. pKG-EZR (GST-EZR1–586(P4T,N6S)) and pKG-EZRn (GST-EZR1–310(P4T,N6S)) were constructed by replacing the moesinNcoI–NcoI or NcoI–SalI fragment in pKG-MSN with the human ezrinNcoI–NcoI or NcoI–SalI (from pAS1-EZRn)2 fragments, respectively. pG3X-EZRc (GST-EZR280–586) was made by inserting the ezrin C-terminalBamHI–BamHI fragment into pGEX-3X vector. pKG-RDX (GST-RDX1–583) was as described by Pestonjamasp et al. (20Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar). pKG-RDXn (GST-RDX1–449) was generated by deleting the pig radixin C-terminal HindIII–HindIII fragment from pKG-RDX and recircularizing the plasmid. pKG-RDXc (GST-RDX373–583) was constructed by subcloning the C-terminalXbaI–XhoI fragment from pKG-RDX into pGEX-KG. PGEX-NF2 (GST-merlin) and pGEX-NFc (GST-merlin C-terminal region, residues 254–595) were as described by Huang et al.(23Huang L. Ichimaru E. Pestonjamasp K. Cui X. Nakamura H. Lo G.Y.H. Lin F.I.K. Luna E.J. Furthmayr H. Biochem. Biophys. Res. Commun. 1998; 248: 548-553Crossref PubMed Scopus (37) Google Scholar). To confirm the correct sequence, all PCR-derived mutants were sequenced using the ABI PRISM DYE Terminator cycle sequencing system (Perkin-Elmer). Wild type and mutated moesin cDNAs were subcloned into derivatives of pET vectors (Stratagene) carrying a T7 promoter for expression of untagged moesin proteins in Escherichia coli and in vitro (41Blacklow S. Kim P. Nat. Struct. Biol. 1996; 3: 758-762Crossref PubMed Scopus (107) Google Scholar). The NdeI–HindIII fragment from pGhuMo, pKG-MSNT558D, and pKG-MSNΔK5 was cloned into pETNde to generate MSN/pET, MSNT558D/pET, and MSNΔK5/pET, respectively. The longerNdeI–HindIII fragment, obtained from pKG-MSNT558A by partial digest with HindIII, was cloned into pETNde to generate MSNT558A/pET. The shorterNdeI–HindIII fragment was cloned into pETNde to make MSNΔC19. The NcoI–NcoI fragment from UIII was cloned into pETNco to generate MSNΔN11/pET. TheEcoRI–XhoI fragment encoding full-length moesin from pGhuMo was subcloned into pCR3 (InVitrogen) to generate MSN/pCR3. The C-terminal PstI–XhoI fragment from pGhuMo was subcloned into pCR3 to make MSNc/pCR3 encoding residues 421–577 with codon 421 as the initiation methionine. Likewise, thePstI–XhoI fragment from pKG-MSNT558D was subcloned into pCR3 to generate MSNcT558D/pCR3. MSNn/pCR3 encoding moesin's N-terminal region residues 1–310 was generated by digesting MSNn-GFP/pCR32 with SmaI and EcoRV (to delete GFP) and recircularization. TheNcoI–NcoI fragment from UIII was inserted into pAS1 and pACT2 to create pAS1-MSNΔn11 (G4BD-HA-moesin12–577) and pACT2-MSNΔn11 (G4AD-HA-moesin12–577), respectively. pAS1-MSNΔn11 and pACT2-MSNΔn11 were digested with SmaI to delete the C-terminal domain and recircularized to generate pAS1-MSNnΔ11 (G4BD-HA-moesinN12–310) and pACT2-MSNnΔ (G4AD-HA-moesinN12–310), respectively. pACT2-MSN (G4AD-HA-moesin) and pACT2-MSNT558D (G4AD-HA-moesin T558D mutant) were constructed by subcloning into pACT2 theEcoRI–XhoI fragment from pGhuMo and pKG-MSNT558D, respectively. pACT2-MSNc (G4AD-HA-moesin C-(404–577)) and pACT2-MSNcT558D (G4AD-HA-moesin C-(404–577/T558D)) were generated by subcloning theEcoRI–XhoI fragment from MSNc/pCR3 and MSNcT558D/pCR3 into pACT2. pACT2-MSNcΔK5 (G4AD-HA-moesin C/KYKTL deleted) and pACT2-MSNcΔc19 (G4AD-HA-moesin C-(1–558)/19 residues truncated) were constructed by subcloning the EcoRI–XhoI fragment from GFP-MSNcΔK5/pCR3 and GFP-MSNcΔc19 into pACT2.2 pAS1-MSN (G4BD-moesin) was constructed by subcloning theBamHI–XhoI fragment, excised from pACT2-MSN by partial digest with BamHI and XhoI into pAS1-NF2 (23Huang L. Ichimaru E. Pestonjamasp K. Cui X. Nakamura H. Lo G.Y.H. Lin F.I.K. Luna E.J. Furthmayr H. Biochem. Biophys. Res. Commun. 1998; 248: 548-553Crossref PubMed Scopus (37) Google Scholar), and predigested with BamHI and SalI (to delete NF2). pAS1-MSNT558D (G4BD-moesin T558D mutant) was created by replacing theNdeI–SalI fragment in pAS1-MSN with theNdeI–SalI fragment from pKG-MSNT558D. pAS1-MSNc (G4BD-moesin C-(404–577)) was made by subcloning theBamHI–XhoI fragment from pACT2-MSNc into pAS1-NF2 predigested with BamHI and SalI (to delete NF2). pACT2-MSNn (G4AD-HA-moesinN1–310) was generated by subcloning theEcoRI–SalI fragment from pAS1-MSNn into pACT2 cut with EcoRI and XhoI. pAS1-MSNn (G4BD-HA-moesin N-terminal region 1–310) was constructed by subcloning the SmaI–SmaI fragment from pACT2-MSN into pAS1. It was not suitable for the study because of high background signal in yeast two-hybrid assays. GST-moesin fusions were expressed in E. colitransformed with corresponding plasmids according to the instructions (Pharmacia Biotech Biotech). Untagged moesin proteins were expressed inE. coli strain BL21(DE3) transformed with pET plasmids carrying moesin inserts according to instructions (Stratagene, Palo Alto, CA). The recombinant moesin proteins were analyzed by SDS-PAGE, Coomassie staining, and Western blot as described previously using rabbit polyclonal antibody pAs90–7 (8Amieva M.R. Furthmayr H. Exp. Cell Res. 1995; 219: 180-196Crossref PubMed Scopus (132) Google Scholar) to verify the expression of desired proteins. Before use for F-actin blot overlay, the recombinant proteins were quantitated by Western blot followed by densitometry and using known amounts of moesin to construct a standard curve. Protein samples were resolved by SDS-PAGE under reducing conditions and electrotransferred to nitrocellulose membranes. The blots were incubated in TTBS (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.05% Tween 20) containing 0.005% sodium azide overnight at 4 °C and in blocking buffer (TTBS containing 5% dry milk and 0.005% sodium azide) overnight at 4 °C. After washing with TTBS, the blots were probed with [α-32P]ATP- or [α-35S]ATP-labeled F-actin (27Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (265) Google Scholar). A. Spudich (Department of Biochemistry, Stanford University) generously supplied rabbit skeletal muscle actin. The probe was prepared as follows. 66 μm G-actin was gel-filtered into G buffer (2 mm Tris-HCl, pH 8.0, 50 μmCaCl2, 0.5 mm dithiothreitol, 50 μm NaN3) to reduce the concentration of free ATP and then incubated for 120 min at room temperature with 125 μCi ([α-32P]ATP at 800 Ci/mm, or [α-35S]ATP at >1000 Ci/mm). Actin polymerization was initiated by the addition of 0.1 volume of 0.5m KCl, 20 mm MgCl2, 0.5 mm CaCl2 (10× F buffer) and continued for 20 min at room temperature, after which F-actin was sedimented at 100,000 × g for 30 min. An estimated 70% of the isotope was incorporated into the F-actin pellet. The F-actin was resuspended at 20 μg/ml in F buffer containing 5 mmphalloidin and 1 mm dithiothreitol. For blot overlay, the protein was resuspended at 20 μg/ml in Western blocking buffer (TTBS; 150 mm NaCl, 20 mm Tris-HCl, pH 8.0, 0.2% Tween 20, 5% fat-free milk powder) containing 5 μmphalloidin and 1 mm dithiothreitol and incubated with preblocked Western blots for 2 h at room temperature. Blots were washed in TTBS four times for 5 min at room temperature and then exposed to film at −80 °C. Co-sedimentation ofin vitro translated, 35S-labeled moesin with polymerized F-actin was based on reported methods (42Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Crossref PubMed Scopus (60) Google Scholar, 43Casella J.F. Torres M.A. J. Biol. Chem. 1994; 269: 6992-6998Abstract Full Text PDF PubMed Google Scholar, 44Van Etten R.A. Jackson P.K. Baltimore D. Sanders M.C. Matsudeira P.T. Janmey P.A. J. Cell Biol. 1994; 124: 325-340Crossref PubMed Scopus (237) Google Scholar) and done with modifications. Phosphatidylinositol (PI) or phosphatidylinositol 4,5-diphosphate (PIP2) (Sigma) was dissolved in distilled water to a final concentration of 1 mg/ml and sonicated three times each for 10 s. In vitro translated proteins were incubated with or without 50 μg/ml PI or PIP2 in polymerization buffer (100 mm KCl, 2 mmMgCl2, 0.2 mm CaCl2, 0.2 mm ATP, 1 mm dithiothreitol, 0.01% NaN3, 5 mm Tris-HCl, pH 8.0) containing 0.75% Triton X-100 for 10 min and centrifuged at 150,000 × gfor 30 min in a Beckman TL-100 ultracentrifuge using Ultra-Clear centrifuge tubes (5 × 41 mm) and a TLA-100.3 rotor to clear protein aggregates. The supernatants were incubated with or without 0.5 mg/ml F-actin in polymerization buffer containing 0.75% Triton X-100 for 30 min, transferred into centrifuge tubes with or without underlying 20% sucrose in polymerization buffer, and centrifuged again as above. Aliquots of the supernatants and pellets that had been rinsed with polymerization buffer were solubilized by boiling in SDS sample buffer, analyzed by SDS-PAGE and autoradiography, and the35S-labeled protein bands were quantitated by densitometry. Identical results were obtained with or without the 20% sucrose cushion, and those without are presented. pET or pCR3 plasmids carrying moesin inserts were transcribed and translatedin vitro using a TNT coupled rabbit reticulocyte lysate system (Promega, Madison, WI) in the presence of T7 RNA polymerase andl-[35S]methionine according to the manufacturer's instructions. In vitro translation products with or without 50 μg/ml PI or PIP2 were incubated with GST fusion proteins, which had been immobilized on glutathione-Sepharose beads and washed four times with phosphate-buffered saline, in binding buffer (200 mm NaCl, 20 mm Tris-HCl, pH 8, 0.2% Triton X-100) at 4 °C. After 1.5 h, the beads were washed six times in binding buffer. Bound proteins were boiled in SDS sample buffer, resolved by SDS-PAGE, and subjected to autoradiography. Pairs of hybrid plasmids were transformed (45Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1762) Google Scholar) into the yeast reporter strain Y190 (46Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar), and co-transformants were selected on SC media lacking Trp and Leu at 30 °C for 4 days. Single colonies were patched onto selective plates, incubated at 30 °C for 3 days, and subjected to a filter lift assay for β-galactosidase activity (46Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar, 47Breeden L. Nasmyth K. Cold Spring Harbor Symp. Quant. Biol. 1985; 50: 643-650Crossref PubMed Scopus (467) Google Scholar) and onto selective plates lacking His and containing 25 mm3-amino-1,2,4-triazol. Incubation was done at 30 °C for 7 days to assay for growth (46Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar). While results of both assays were in good agreement, those of the β-galactosidase assay are presented. As a first step, a series of mutations of Thr558 and flanking sequences were introduced into moesin by polymerase chain reaction, and the mutants were subcloned to yield GST fusion proteins. Mutant and wild-type GST-moesin were then expressed in E. coli, and effects of the mutation on F-actin binding were examined by F-actin blot overlay with 32P- (27Mackay D.J.G. Esch F. Furthmayr H" @default.
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• W1977917516 title "Replacement of Threonine 558, a Critical Site of Phosphorylation of Moesin in Vivo, with Aspartate Activates F-actin Binding of Moesin" @default.
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